Inflammatory leukocytes synthesize a number of inflammatory mediators that are isolated intracellularly and stored in cytoplasmic membrane-bound granules. Examples of such mediators include, but are not limited to, myeloperoxidase [MPO] in neutrophils (see, for example, Borregaard N, Cowland J B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89:3503-3521), eosinophil peroxidase [EPO] and major basic protein [MBP] in eosinophils (see, for example, Gleich G J. Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol 2000; 105:651-663), lysozyme in monocytes/macrophages (see, for example, Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on the TPA-induced monocytic differentiation. J Leukoc Biol 1992; 52:173-182; and Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J Immunol 2003; 170:5276-5280), and granzyme in natural killer (NK) cells and cytotoxic lymphocytes (see, for example, Bochan M R, Goebel W S, Brahmi Z. Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability. Cell Immunol 1995; 164:234-239; Gong J H., Maki G, Klingemann H G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann H G, Martinson J A, Tam Y K. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematother Stem Cell Res 2001; 10:369-383; and Takayama H, Trenn G, Sitkovsky M V. A novel cytotoxic T lymphocyte activation assay. J Immunol Methods 1987; 104:183-190). Such mediators are released at sites of injury and contribute to inflammation and tissue repair such as in the lung and elsewhere. It is known that leukocytes release these granules via an exocytotic mechanism (see, for example, Burgoyne R D, Morgan A. Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; and Logan M R, Odemuyiwa S O, Moqbel R. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion. J Allergy Clin Immunol 2003; 111: 923-932), but regulatory molecules and specific pathways involved in the exocytotic process have not been fully described.
Several exogenous stimuli can provoke degranulation of leukocytes via a pathway that involves activation of protein kinase C and subsequent phosphorylation events (see, for example, Burgoyne R D, Morgan A. Secretory granule exocytosis. Physiol Rev 2003; 83:581-632; Logan M R, Odemuyiwa S O, Moqbel R. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion. J Allergy Clin Immunol 2003; 111: 923-932; Smolen J E, Sandborg R R. Ca2+-induced secretion by electropermeabilized human neutrophils: the roles of Ca2+, nucleotides and protein kinase C. Biochim Biophys Acta 1990; 1052:133-142; Niessen H W, Verhoeven A J. Role of protein phosphorylation in the degranulation of electropermeabilized human neutrophils. Biochim. Biophys. Acta 1994; 1223:267-273; and Naucler C, Grinstein S, Sundler R., Tapper H. Signaling to localized degranulation in neutrophils adherent to immune complexes. J Leukoc Biol 2002; 71:701-710).
MARKS protein (where MARCKS as used herein means “Myristoylated Alanine-Rich C Kinase Substrate”), is a ubiquitous phosphorylation target of protein kinase C (PKC), and is highly expressed in leukocytes (see, for example, Aderem A A, Albert K A, Keum M M, Wang J K, Greengard P, Cohn Z A. Stimulus-dependent myristoylation of a major substrate for protein kinase C. Nature 1988; 332:362-364; Thelen M, Rosen A, Nairn A C, Aderem A. Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane. Nature 1991; 351:320-322; and Hartwig J H, Thelen M, Rosen A, Janmey P A, Nairn A C, Aderem A. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992; 356:618-622. MARCKS protein is mechanistically involved in a process of exocytotic secretion of mucin by goblet cells that line respiratory airways (see, for example, Li et al., J Biol Chem 2001; 276:40982-40990; and Singer et al., Nat Med 2004; 10:193-196). MARCKS is myristoylated via an amide bond at the N-terminal amino acid in the MARCKS protein's amino acid sequence at the alpha-amine position of the glycine which resides at the N-terminus (i.e., at position 1) of amino acid sequence. In airway epithelial cells, the myristoylated N-terminal region of MARCKS appears to be integral to the secretory process. By the N-terminus of the MARCKS protein is meant the MANS peptide which contains Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1), which are L-amino acids. Additionally, the peptide fragments of the MANS peptide disclosed herein, also preferably are composed of L-amino acids. The mechanism appears to involve binding of MARCKS, a myristoylated protein, to membranes of intracellular granules.
An N-terminal myristoylated peptide from the N-terminus of MARCKS has been shown to block both mucin secretion and binding of MARCKS to mucin granule membranes in goblet cells (see, for example, Singer et al., Nat Med 2004; 10:193-196). This peptide contains 24 amino acids of the MARCKS protein beginning with the N-terminal glycine of the MARCKS protein which is myristoylated via an amide bond and is known as myristoylated alpha-N-terminal sequence (MANS); i.e., Myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO: 1). Also Vergeres et al., J. Biochem. 1998, 330; 5-11, discloses that the N-terminal glycine residue of MARCKS proteins is myristoylated via a reaction catalyzed by myristoyl CoA:protein N-myristoyl transferase (NMT).
In inflammatory diseases, such as asthma, COPD and chronic bronchitis; in genetic diseases such as cystic fibrosis; in allergic conditions (atopy, allergic inflammation); in bronchiectasis; and in a number of acute, infectious respiratory illnesses such as pneumonia, rhinitis, influenza or the common cold, arthritis or auto-immune diseases, inflammatory cells are usually found in or migrate to areas of injury or infection associated with inflammatory disease states, especially in or to respiratory passages or airways of patients suffering from such diseases. These inflammatory cells can contribute greatly to the pathology of diseases via tissue damage done by inflammatory mediators released from these cells. One example of such tissue damage or destruction via this chronic inflammation occurs in cystic fibrosis patients where mediators released from neutrophils (e.g., myeloperoxidase [MPO]) induce the desquamation of the airway epithelial tissue.
MARCKS, a protein of approximately 82 kD, has three evolutionarily-conserved regions (Aderem et al., Nature 1988; 332:362-364; Thelen et al., Nature 1991; 351:320-322; Hartwig et al., Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996; 271:18797-18802): an N-terminus, a phosphorylation site domain (or PSD), and a multiple homology 2 (MH2) domain. Human MARCKS cDNA and protein is known and reported by Harlan et al., J. Biol. Chem. 1991, 266:14399 (GenBank Accession No. M68956) and also by Sakai et al., Genomics 1992, 14: 175. These sequences are also provided in a WO 00/50062, which is incorporated in its entirety by reference. The N-terminus, an alpha-amino acid sequence comprising 24 amino acid residues with a myristic acid moiety attached via an amide bond to the N-terminal glycine residue is involved in binding of MARCKS to membranes in cells (Seykora et al., J Biol Chem 1996; 271:18797-18802) and possibly to calmodulin (Matsubara et al., J Biol Chem 2003; 278:48898-48902). This 24 amino acid sequence is known as the MANS peptide.